Computational and Chemical Biology, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy.
Electron Microscopy Facility, Istituto Italiano di Tecnologia, Via Morego, 30, 16163 Genoa, Italy.
Int J Mol Sci. 2022 Jul 28;23(15):8338. doi: 10.3390/ijms23158338.
The cytotoxic action of anticancer drugs can be potentiated by inhibiting DNA repair mechanisms. RAD51 is a crucial protein for genomic stability due to its critical role in the homologous recombination (HR) pathway. BRCA2 assists RAD51 fibrillation and defibrillation in the cytoplasm and nucleus and assists its nuclear transport. BRC4 is a peptide derived from the fourth BRC repeat of BRCA2, and it lacks the nuclear localization sequence. Here, we used BRC4 to (i) reverse RAD51 fibrillation; (ii) avoid the nuclear transport of RAD51; and (iii) inhibit HR and enhance the efficacy of chemotherapeutic treatments. Specifically, using static and dynamic light scattering, transmission electron microscopy, and microscale thermophoresis, we show that BRC4 eroded RAD51 fibrils from their termini through a "domino" mechanism and yielded monomeric RAD51 with a cumulative nanomolar affinity. Using cellular assays (BxPC-3, pancreatic cancer), we show that a myristoylated BRC4 (designed for a more efficient cell entry) abolished the formation of nuclear RAD51 foci. The present study provides a molecular description of RAD51 defibrillation, an essential step in BRCA2-mediated homologous recombination and DNA repair.
抗癌药物的细胞毒性作用可以通过抑制 DNA 修复机制来增强。RAD51 是基因组稳定性的关键蛋白，因为它在同源重组 (HR) 途径中起着至关重要的作用。BRCA2 协助 RAD51 在细胞质和核中的纤颤和解纤颤，并协助其核转运。BRC4 是源自 BRCA2 第四个 BRC 重复的肽，它缺乏核定位序列。在这里，我们使用 BRC4 来：(i) 逆转 RAD51 纤颤；(ii) 避免 RAD51 的核转运；和 (iii) 抑制 HR 并增强化疗治疗的效果。具体来说，我们使用静态和动态光散射、透射电子显微镜和微尺度热泳动，表明 BRC4 通过“多米诺骨牌”机制从 RAD51 纤颤的末端侵蚀 RAD51 纤颤，并产生具有累积纳摩尔亲和力的单体 RAD51。使用细胞测定法（BxPC-3，胰腺癌），我们表明，一种豆蔻酰化的 BRC4（设计用于更有效的细胞进入）消除了核 RAD51 焦点的形成。本研究提供了 RAD51 解纤颤的分子描述，这是 BRCA2 介导的同源重组和 DNA 修复的一个基本步骤。
